Despite recent progress, it remains unclear how progranulin deficiency leads to development of frontotemporal lobar degeneration (FTLD) and contributes to Alzheimer?s Disease (AD). Progranulin is cleaved into bioactive granulin peptides that functionally oppose the progranulin holoprotein. Many believe that progranulin haploinsufficiency equally depletes progranulin and granulin levels, yet this has never been directly measured. Fundamental knowledge gaps also exist regarding proteases that cleave progranulin to produce granulins, the normal function of these cleaved peptides and consequences of granulins on protein homeostasis and disease. The long-term goal of this research is to understand how age-related changes in protein homeostasis affect risk of neurodegenerative diseases like AD and FTLD. The objective of this application is to understand the production, function and consequences of granulins using C. elegans, cultured cells and patient-derived biospecimens. The central hypothesis is that age and physiological stress promote the liberation of granulins in the endolysosomal system, where granulins bind to and inhibit specific lysosomal proteases, ultimately impairing neuronal protein homeostasis. This hypothesis is based on extensive preliminary data produced by the applicant that granulin production increases with age and stress, granulins promote TDP-43 accumulation and enhance its toxicity, and these cysteine-rich peptides can bind and sterically inhibit the active site of a lysosomal aspartyl protease, cathepsin D. The rationale for this work is that understanding production and function of both progranulin and granulins is critical to safely targeting these molecules in the treatment of FTLD and other diseases. The central hypothesis will be tested through three specific aims: 1) understand the regulated production of granulins, 2) determine the specificity profile of cathepsin inhibition by granulins, and 3) elucidate the effects of granulins on protein homeostasis. The proposed research is conceptually innovative because it seeks to directly implicate age-associated granulin accumulation, rather than or in addition to progranulin deficiency, as a driving force in neurodegeneration related to progranulin mutations. It is also technically innovative because of development of a novel fluorescent progranulin cleavage sensor tool, use of a new set of anti-human granulin antibodies and application of BioLayer Interferometry (BLI) for label-free, real- time monitoring of granulin/cathepsin interaction kinetics. The proposed research will contribute essential information about the production, specificity and functional consequences of granulins. This contribution is significant because it will improve understanding of how progranulin haploinsufficiency leads to FTLD, provide fundamental new knowledge regarding the normal regulation of lysosomal proteases and lay the foundation for development of safe progranulin replacement therapies.